Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a "real" 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength.
There's also one quite strange thing about the nanodrop - they sell the "calibration check solution" (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it's off to some certain extend: But then you're stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration-option into the software, but at the moment the instrument tells you "calibration failure" and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them with locktite. Anyway, at least for our mid to high concentrated samples the nanodrop is not showing large fluctuations so we are happy with it. But everyone using a nanodrop should check it from time to time - as I found out that ours was off more than 20% at one day - which raised some trouble of course. Cheers, Gregor Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Filip Van Petegem Gesendet: Donnerstag, 4. Dezember 2008 22:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] suggestions for UV spectrometer I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll <[EMAIL PROTECTED]> wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules "shadowing" one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... ---------------------------------------------------------------------------- ----------- Patrick J. Loll, Ph. D. Professor of Biochemistry & Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
